Int.J.Curr.Microbiol.App.Sci (2013) 2(10):506-515
ISSN: 2319-7706 Volume 2 Number 10 (2013) pp. 506-515
http://www.ijcmas.com
Original Research Article
Wet-heat inactivation of bacterial endospores in packaged fruit juices
A.A.Brooks*
Department of Microbiology, University of Calabar, Nigeria
*Corresponding author
ABSTRACT
Keywords
Bacterial
endospores;
Inactivation;
Fruit juices;
Thermal
resistance.
Thermal resistance of bacterial endospores was evaluated at different temperature
regimes using wet-heat inactivation process. This was with the view to determining
the thermal inactivation characteristics of bacterial spores recovered from selected
packaged fruit juices. The bacterial strains recovered were Bacillus subtilis K16,
Bacillus coagulans B74, Bacillus cereus Y28B, Clostridium perfingens T20 and
Clostridium sporogenes A63. The success of the inactivation process was
determined by the ability of the inactivated spores to germinate and form colonies.
The decimal reduction times (D - values) for spores from strains K16, B74, Y28B, T20
and A63 were 2.20, 1.78, 2.32, 2.16 and 2.0 minutes at 650C, 750C, 850C and 950C
respectively. The Z values were 7.40C, 8.50C, 6.80C, 7.60C and 8.70C respectively.
There were high variations in the heat resistance amongst the five spore crops
studied, in the order B74>T20> A63>K16>Y28B. There was a perfect correlation
between the process temperature and the D-values with high correlation
coefficients. The linear regression analysis of the data for all the strains fitted into
the linear model Y=a + b x. More than 75.0% of the spores from Y28B were
inactivated within 30 minutes of heat treatment at 650C. Spores from other strains
exhibited marked heat resistance at the different temperature regimes tested.
Surviving sub population of spores were observed after heat treatment for 30
minutes at all temperatures tested. Therefore, temperature higher than 950C may be
required to inactivate bacterial spores in fruit juices.
Introduction
A wide variety of fruit juices are produced
and consumed globally. Fruit juices are
rich in vitamins and they supply the
human body with essential precursors for
effective metabolic processes. They are
usually consumed without much regard to
their microbiological status and safety
because fruit juices are not often
considered as high risk products. Although
some of the fruit juices available in world
506
markets are terminally pasteurized and are
therefore expected to contain no microbial
contaminant, quite a number of them are
either not subjected to thermal and other
conventional
treatments
or
are
inadequately treated. Such untreated or
inadequately treated products may be
microbiologically unfit for drinking as
they may contain pathogenic bacteria such
as Escherichia coli 0157:H7 and
Int.J.Curr.Microbiol.App.Sci (2013) 2(10):506-515
Salmonella species that can cause serious
illnesses (Walner et al., 2006). Some of
these bacteria can be quite resistant in
acidic conditions and survive for a long
period in acidic environment provided by
the juices.
Their germination and outgrowth often
cause substantial problems such as
spoilage, lower quality and potential risk
of food poisoning outbreaks among
consumers. Fruit juices have been reported
to be contaminated by spore forming
bacteria Alicyclobacillus acidoterrestris
(Silva et al., 2012) and Bacillius anthracis
(Leishman et al., 2010). Bacterial spores
are more resistant to heat and other
preservation treatments than the vegetative
cells. Fruits juice industries still rely
largely on the use of preservative and or
thermal treatment such as pasteurization
and ultra high temperature (UHT)
processes to produce safe fruit drinks.
According to Mustafa et al. (2010), these
processes depend on both temperature of
exposure and the time required to
accomplish the required destruction rate.
In order to preserve the nutritional and
organoleptic qualities of the fruit juices,
the heat treatment period is usually brief
and the time is not enough to kill a good
proportion of the bacterial spores.
The results of the investigation conducted
by Safe Food Rapid Response Network in
Colorado State University, USA, revealed
that some cases of out breaks of food
borne illnesses in USA were linked to the
consumption of unpasteurized fruit juices.
For example, the 1996 multi state outbreak
of E. coli infection in USA due to the
consumption of untreated apple juice; an
outbreak of Cryptosporidium infection in
2003 due to the drinking of inadequately
treated apple cider and the outbreak of
Salmonella infection in 2005 as a result of
the consumption of inadequately treated
orange juice labeled as fresh squeezed.
In Nigeria cases of food borne illnesses
arising from the consumption of
contaminated fruit juices abound but there
is no reliable epidemiological estimate. To
date, no precise and consistent data exists,
hence it is difficult to assess the impact of
fruit juice related illnesses on the Nigerian
population. However, the country over the
years has been grappled with the problem
of food borne illnesses with their attendant
social, economic and health implications
(Ifenkwe, 2012).
Inactivation of bacterial spores required
high temperature and long heating time.
Although spore dormancy and associated
resistance are very stable, these properties
are easily lost during inactivation.
Therefore, proper spore inactivation is
essential for the production of safe and
quality fruit juices. Silva et al. (2012)
reported on bacterial spore inactivation at
45oc using high pressure processing while
Ramirez-lopez (2006) reported on wet heat
inactivation of bacterial spores from wheat
offals. There is no report (to the
knowledge of the author) on wet-heat
inactivation of bacterial endospores
recovered from fruit juices sold in Nigeria,
hence this study.
One of the commonest contaminants of
fruit juices are the bacterial endospores,
which usually have access to the drink
from outside the fruits at the
manufacturing stage, especially when such
drinks are produced under unhygienic
conditions. The anoxic environment
provided
by
packaging
favours
germination and growth of the endospores
and liberation of toxins into the juice.
Fruit juices are often sealed and terminally
pasteurized and are supposed to be
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Int.J.Curr.Microbiol.App.Sci (2013) 2(10):506-515
microbiologically stable. However, owing
to high incidence of fruit juice related
illnesses in Nigeria, it is justifiable to
ascertain:
surfaces of the packages were properly
disinfected with methylated spirit.
Contents were properly mixed by shaking
manually. Thereafter the packages were
aseptically opened and 1.0ml of each fruit
juice was used to perform serial dilution in
¼ strength of Ringer solution. One
milliliter of the diluted cells was
inoculated in brain-heart infusion (BHI)
broth and incubated at 55oC for 2-5 days.
Reinforced clostridial medium was
employed for isolation and enumeration of
anaerobic spore-forming bacteria using
pour plate technique, while aerobic spore
formers were enumerated on brain-heart
infusion agar.
If the pasteurization temperature and
exposure time usually employed in fruit
juice industries are enough to adequately
kill the bacterial endospores in the finished
products, and (ii) if the manufacturers of
fruit juices have complied with the
microbiological standards specified by
ICMSF for fruit juices. This study
therefore is aimed at evaluating the
thermal resistance of bacterial spores in
packaged fruit juices by wet-heat
inactivation
process
at
different
temperature regimes.
Identification of spore forming bacteria
The spore forming bacteria isolated from
the fruit juices were identified using the
methods of Oranusi et al. (2012) and Kort
et al., (2005), assisted by the identification
scheme outlined in Bergey s Manual of
Systematic Bacteriology (Holt et al.,
1994).
Materials and Methods
Sample collections
Fifty samples of fruit juices, comprising
ten each from different packaged Chivita,
Five alive, Dansa. Tampico and Frutta
were purchased randomly from different
super markets across Southern Nigeria.
The packages of the fruit juices were
visually examined for any defect such as
bloating, leakage and physical damage.
Other information on the packages
recorded included manufacture and expiry
dates, batch numbers, National Agency for
Food and Drug Administration and
Control
(NAFDAC)
numbers,
manufacturers addresses, preservatives
used and composition of the fruit juices.
The samples were properly labeled and
taken to the laboratory for analysis.
Preparation of bacterial endospores
About 250ml of broth cultures of the
different spore forming bacteria were
prepared in 500ml-shake flasks at 150 rpm
at 37oC. The flasks contained mineral salt
medium initially described by Neidheart et
al., (1974) with some modifications. The
pH of the medium was adjusted to 7.6 with
1N KOH. Spores were harvested after 4
days of incubation at 37oC and purified by
washing repeatedly in sterile distilled
water, each time by centrifugation at
x1000g for 30 minutes. The supernatant
obtained was heated for 10 minutes at
65oC to eliminate any vegetative cell. The
purity of spore sample was confirmed
visually and by microscopic examination
after spore staining.
Sample Analysis
Screening the fruit juices for bacterial
contaminants
Before the samples were screened, the
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Int.J.Curr.Microbiol.App.Sci (2013) 2(10):506-515
The number of spores in the cell-free spore
suspension was determined using Nabeaur
hemocytometer followed by serial dilution
in sterile peptone salt broth to obtain
different spore concentrations ranging
from 0.2x106 to 4.0x106 spores per ml.
The final spore concentrations were stored
in peptone salt broth at 4oC.
experimental plates. After incubation
visible colonies on the plates were counted
and the results compared with the total
number of spores counted in the
hemocytometer.
Inactivation of the spores
The D-values (time to inactivate 90% of
the spore population) were determined
from the straight line portion of the
survival-time curves by plotting log of
survival counts against their corresponding
heating times. The values were obtained
from the survival curve equation
Determination of Decimal Reduction
Time (D-Values) of Spores
The bacterial spores were inactivated by
wet-heat using the screw-cap tube method
described by the Koolman (1973). In this
method, 200µl of the different spore
concentrations was injected separately
with a Hamilton syringe into metal screwcap tube which were preheated at 60oC for
10 minutes for equilibration. The tube
contained 9.8ml of trypticase soy broth as
inactivation medium Heating was carried
out. With the tubes completely immersed
in a glycerol broth and heated up to 95oC
for 30minutes. There after the spore
suspensions were diluted 10 times with
sterile distilled water and counted using of
Nebeaur hemocytometer. The success of
the inactivation process was determined by
their ability to germinate on brain heart
infusion agar and formed colonies.
D=
t
LogN logN0
Where N is the final spore population, t is
the time at LogN and N0 is the initial
population of the straight line portion on
the destruction curve produced by plotting
the log10 numbers of survivors against
time at 1000C (Harrigan, 1998).
Determination of Viability of the Spores
The viability of spores was determined by
inoculating the inactivated spores in
trypticase soy agar and incubating at 37oC
for 4 days. Serial dilution of the
inactivated spore suspension was carried
out in peptone-saline solution (0.1%
peptone, 0.85% Nacl). One milliliter each
of the diluted spore suspension was added
separately in duplicate to trypticase soy
agar by pour plate technique and incubated
for 4 days. The number of visible colonies
on the plates was counted thereafter.
Effect of different temperature regimes
on spore inactivation
Heat-treated spore suspensions from the
different strains containing known
population of bacterial spores were
incubated in trypticase soy broth at 650C,
750C, 850C and 950C for 30minute
respectively. Thereafter the spores were
serially diluted in sterile distilled water,
plated on brain heart infusion (BHI) agar
and incubated at 370C for up to 4 days.
Control experiments consisted of BHI agar
plates inoculated with similar spore stains
without heat treatment and incubated as in
Effect of exposure time on spore
viability
Known population of the five spore crops
obtained from the different strains of
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Int.J.Curr.Microbiol.App.Sci (2013) 2(10):506-515
bacterial contaminants were heated at
650C for ten to seventy minutes.
Thereafter 1.0ml of the heated spore
suspensions were placed on BHI agar and
reinforced clostridial media respectively.
The plates were incubated at 370C for 4
days, after which the surviving colonies
were determined and log numbers of the
surviving colonies were plotted against
exposure time at the test temperature.
in thermal resistance of the various strains
studied
in
the
order
B74>T20>
A63>K16>Y28B. The results 18.5% of
strain B74 was inactivated within 30
minutes of heat treatment. Spores from
other strains also exhibited marked
resistance at this temperature.
A total of 73.3% of spores from strain
Y28B germinated at 650C and subsequently
became inactivated within 30 minutes at
higher temperature. This pronounced
inactivation may be due to the fact that
low inactivation temperatures usually
activate spores allowing increased
germination of spores. This assertion
agrees with an earlier report by Kort et al.
(2005). The percentage of viable spores
under the same conditions for strains B74,
T20, A63, and K16 were 81.5%,
68.0%,62.5% and 51.7% respectively.
The low degrees of germination of spores
from these strains at 650C were expected
because the spores were isolated from heat
resistant strains. Moreso, at pasteurization
temperature, the spores may be in deep
dormancy and can only be activated at
high temperature (Iciek et al., 2005).
Statistical analysis
The data obtained from this study were
analyzed by Analysis of Variance
(ANOVA) and correlation analysis,
according to Sokal and Rholf (1983), to
determine the significant differences
amongst the treatments.
Results and Discussion
All the packaged fruit juices examined
yielded moderate growth of spore forming
bacteria. A total of five spore crops were
isolated from the different bacterial strains
recovered from the fruit juices. The
bacterial strains were Bacillus subtilis K16,
Bacillus coagulans B74, Bacillus cereus
Y28B, Clostridium perfringens T20 and
Clostridium sporogenes A63.
When these spore crops were incubated at
950C for 30 minutes, the percentage spore
inactivation for strains B74, T20, A63 , K16
and Y28B increased to 64.4%, 69.0%,
71.3% , 72.7% and 94.1%, respectively.
Surviving spores after heat treatment were
observed at all temperatures tested. The
viable spore subpopulations observed at all
temperatures might be due to the fact that
fruit juices have high moisture content.
Therefore the high moisture content might
have offered some protection against
thermal
inactivation
of
bacterial
endospores. The variation in the
percentage inactivation of spores subjected
to different degrees of thermal treatment
might also be due to differences in the heat
Microscopic examination of all the
thermally
treated
spores
revealed
pronounced morphological malformations
such as shrinkage and ridge on the outer
spore coat. Similar morphological changes
were observed by Mustafa et al., (2010)
during their study on morphological
changes induced by wet-heat in Bacillus
cereus endospores.
The inactivation curves of endospores
from bacterial strains studied at different
temperatures are shown in Figures 1 (a)
and (b). The result shows high variations
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Int.J.Curr.Microbiol.App.Sci (2013) 2(10):506-515
resistant capabilities of the different strains
studied.
Table.1 Z-values of bacterial endospores
recovered from packaged fruit juices.
Bacterial Strain
Z- values
(0C )
Bacillus subtilis K16
7.4
Bacillus coagulans B17
8.5
Bacillus cereus Y28B
6.8
Clostridium perfringens 7.6
T20
Clostridium sporogenes 8.7
A63
In this study, the thermal inactivation of
heat resistant bacterial spores did not
follow first-order kinetics, because some
spores were more heat resistant than
others. This is evidenced by the tailing in
Figures 1(a) and (b), due to resistant
subpopulation. The most resistant strain
encountered in this study was Bacillus
coagulans B74, with only 64.4%
inactivation at 950C after 30 minutes of
heat treatment.
Figure 3 shows the survivor - time curve
for the thermal resistant spores at 850C and
950C respectively. Each of the survivor
curve shows a clear tailing deviation,
which implies that the wet heat
inactivation of the bacterial endospores
has a positive correlation with the
temperature of exposure. The result also
shows that about 35.6 % of strain B74 was
still viable after 30 minutes exposure at
950C. Strains Y28B, K16, T20 and A63
exhibited 5.9 %, 27.3%, 31.0% and 28.7%
viability at this temperature respectively.
Beyond 30 minutes exposure time, the
percentage spore viability reduced
drastically toward zero.
The decimal reduction times (D-values)
for spores from the different strains
studied at different temperatures are
depicted in Figures 2 (a) and (b), while the
Z values are shown in Table 1. There was
a perfect correlation between the process
temperature and the D-values with high
correlation coefficients. The linear
regression analysis of the data for all the
strains fitted into the linear model Y=a + b
x.
This perfect linearity was expected
because when a spore population is heated,
the process of spore activation and
vegetative cell destruction actually begins
with increase in temperature until a high
enough temperature is reached when a
greater percentage of the population would
be inactivated. A similar observation was
made by Brown (1994) during his study on
spore resistance and ultra heat treatment
process. The Z-values for Bacillus strains
K16, B74 and Y28B were 7.40C, 8.50C and
6.80C. These values are in agreement with
those commonly observed for Bacillus
spores which usually vary between 5.50C
and 100C (Russell, 2003).
In conclusion therefore, temperature
higher than 950C may be required to
inactivate bacterial spores in fruit juices
within 30 minutes. This study has
determined moist heat resistant bacteria
and their inactivation characteristics in
fruit juices. The findings from this
investigation will enable fruit juice
industry to establish appropriate process
controls to ensure that the final product is
free from bacteria that can reduce the
quality of the product. However, since
there are many other factors that affect
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Int.J.Curr.Microbiol.App.Sci (2013) 2(10):506-515
Figure.1a Inactivation curves of strains Y28B and K16
Lcfu
2.5
2
1.5
Y28B
1
0.5
0
60
70
80
90
Process Temperature (0C)
Figure.1b Inactivation curves of strains B74, T20 and A63
Lcfu
2.5
2
B74
T20
1.5
1
0.5
0
60
70
80
Process Temperature (0C)
512
90
A63
K16
Int.J.Curr.Microbiol.App.Sci (2013) 2(10):506-515
Figure.2a Thermal Reduction Times (D values) of bacterial endospores
from strains Y28B and K16.
Log 10 D
3
2.5
Y= 7. 18 + 0.066X
R2 = 0.9820
2
1.5
1
Y28B
K16
0.5
0
65
75
85
Temperature (0C)
Figure.2b Thermal Reduction Times (D values) of bacterial endospores
from strains B74, T20 and A63.
Log 10 D
3.5
3
B74
T20
2.5
2
1.5
1
0.5
0
65
75
85
0
Temperature ( C)
513
95
A63
Int.J.Curr.Microbiol.App.Sci (2013) 2(10):506-515
Lcfu
Figure.3a Survivor-Time curve for thermal resistant spores Y28B, K16, B74,
T20 and A63 at 85°C
Time (Minutes)
Figure.3b Survivor-Time curve for thermal resistant spores Y28B, K16, B74,
T20 and A63 at 95°C
Lcfu
Time (Minutes)
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Int.J.Curr.Microbiol.App.Sci (2013) 2(10):506-515
resistance of bacteria, validation of a
processing method should be undertaken
under realistic conditions using fruit juice
as growth medium.
Koolman, W. J., 1973.The screw cap tube
technique: a new accurate technique
for the determination of the wet heat
resistance of bacterial spores. In: A. N.
Baker, G. W. Gould and J. Wolf (ed.).
Spore Research. Academic Press,
London, United Kingdom, pp 87- 92.
Kort, R., A.C. O Brien, I.H.M. van
Stokkum, S.J.M.
Oomes, W.
Crielaard, K.J. Hellingwerf and Brul,
S. 2005. Assessment of heat resistance
of bacterial spores from food product
isolates by fluorescence monitoring of
dipicolinic acid release. Appl. Environ.
Microbiol. 71(7) :3556-3564.
Leishman, O.N., M.J. Johnson, T.P.
Labuza and Diez-Gonzalez, F. 2010.
Survival of Bacillus anthracis spores
in fruit juices and wine. J. Food
Protect. 73(9): 1694-1697.
Mustafa, N. E. M., U. Keller, V. Malkus,
D. Harmsen, R. Reichelt, A.A. Hussein
and
El-sanousi,
S.M.
2010.
Morphological changes induced by
wet- heat in Bacillus cereus
endospores. Curr.Res.Bacteriol. 3:214226.
Neidhart, F. C., P.I. Bloch and Smith, D.
F. 1974. Culture medium for
enterobacteria. J. Bacteriol. 119:736747
Oranusi, U.S., W. Braide and Osigwe, G.
A. 2012. Investigation on the microbial
profile of canned foods. J. Biol. Food
Sci. Res. 1(1):15-18.
Ramirez
lopez, L. M., 2006. Heat
inactivation
of
thermo-resistant
bacteria isolated from poultry offal.
M.SC thesis presented to the Graduate
School of Clemson University.
References
Beaman, T. C., and Gerhardt, P. 1986.
Heat resistance of bacterial spores
correlated with protoplast dehydration,
mineralization and thermal adaptation.
Appl. Environ. Microbiol. 52:12421246.
Brown, K. L., 1994. Spore resistance and
ultra heat treatment process. J.Appl.
Bacteriol. Sym. Supple.76:67s-80s.
Harrigan, W. F., 1998. Effect of heat on
microorganisms: The determination of
decimal reduction time (D values) and
Z values. Laboratory methods in food
microbiology, San Diego, California,
Academic Press, pp. 123-135.
Holt, J. G., N.P. Krieg, P.H.A. Sneath, J.
Satley and Williams, S. T. 1994.
Bergey s Manual of Determinative
Bacteriology. (9th edn.) Williams and
Wilkins Publishers, Baltimore.pp. 787.
I. C. M. S. F. ,1974. Sampling for
microbiological analysis. International
Commission
on
Microbiological
Specification for Food: Principles and
specific applications, University of
Toronto Press, pp. 1-18.
Iciek, J., A. Papiewska and Molska, M.
2005.
Inactivation
of
Bacillus
stearothermophilus spores during
thermal processing. J. Food Engineer.
171-173
Ifenkwe, G. E., 2012: Food safety
regulations; reducing the risk of foodborne diseases in rural communities of
Abia State, Nigeria .Agricult. Sci. Res.
J. 2(7):384-389.
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